The Journal of Neuroscience, July 28, 2004 • 24(30):6785–6790 • 6785 Behavioral/Systems/Cognitive ␣ Requirement of 5-GABAA Receptors for the Development of Tolerance to the Sedative Action of Diazepam in Mice Carolien van Rijnsoever,1* Marcus Ta¨uber,1* Mohamed Khaled Choulli,1 Ruth Keist,1 Uwe Rudolph,1 Hanns Mohler,1,2 Jean Marc Fritschy,1 and Florence Crestani1 1Institute of Pharmacology and Toxicology, University of Zu¨rich, 8057 Zu¨rich, Switzerland, and 2Department of Applied Biosciences, Swiss Federal Institute of Technology, 8057 Zu¨rich, Switzerland Despite its pharmacological relevance, the mechanism of the development of tolerance to the action of benzodiazepines is essentially ␣ unknown. The acute sedative action of diazepam is mediated via 1-GABAA receptors. Therefore, we tested whether chronic activation of ␣ ␣ ␣ ␣ these receptors by diazepam is sufficient to induce tolerance to its sedative action. Knock-in mice, in which the 1-, 2-, 3-, or 5-GABAA receptors had been rendered insensitive to diazepam by histidine–arginine point mutation, were chronically treated with diazepam (8 d; ⅐ Ϫ1 ⅐ Ϫ1 ␣ ␣ 15 mg kg d ) and tested for motor activity. Wild-type, 2(H101R), and 3(H126R) mice showed a robust diminution of the ␣ ␣ motor-depressant drug action. In contrast, 5(H105R) mice failed to display any sedative tolerance. 1(H101R) mice showed no alter- ation of motor activity with chronic diazepam treatment. Autoradiography with [ 3H]flumazenil revealed no change in benzodiazepine ␣ binding sites. However, a decrease in 5-subunit radioligand binding was detected selectively in the dentate gyrus with specific ligands. This alteration was observed only in diazepam-tolerant animals, indicating that the manifestation of tolerance to the sedative action of ␣ ␣ diazepam is associated with a downregulation of 5-GABAA receptors in the dentate gyrus. Thus, the chronic activation of 5-GABAA ␣ receptors is crucial for the normal development of sedative tolerance to diazepam, which manifests itself in conjunction with 1-GABAA receptors. Key words: diazepam; GABAA receptor; tolerance; motor activity; dentate gyrus; knock-in mice Introduction al., 2001). In particular, a selective decrease in the expression of ␣ ␥ Loss of sedative efficacy of diazepam with chronic treatment has genes encoding the 1- and 2-subunits in dendrites and spines of been proposed to result from the development of adaptive pro- cortical pyramidal cells has been associated with tolerance to the cesses counteracting the repeated enhancement by the benzodi- anticonvulsant action of diazepam (Costa et al., 2002). In addi- ␣ azepine of GABAA receptor-mediated inhibitory neurotransmis- tion, 5-GABAA receptors were affected, as shown by an increase ␣ sion (Steppuhn and Turski, 1993; File and Fernandes, 1994; in 5-subunit mRNA in the frontoparietal cortex or a reduced Fernandes et al., 1996; Marin et al., 1996, 1999; Perez et al., 2003). radioligand binding in the hippocampus after 2 or 3 weeks of Functional alterations of GABAA receptors have frequently been diazepam administration (Impagnatiello et al., 1996; Pesold et al., reported after various chronic treatment regimens with diazepam 1997; Li et al., 2000). (Hutchinson et al., 1996; Itier et al., 1996; Primus et al., 1996; Ali The recognition of distinct pharmacological functions of and Olsen, 2001; Costa et al., 2001; Bateson, 2002). Uncoupling GABAA receptor subtypes opened new avenues to investigate the of the allosteric interaction between the benzodiazepine binding mechanisms of tolerance. Using a histidine-to-arginine point site and the GABA site, probably linked to GABAA receptor in- mutation strategy that selectively abolishes diazepam binding to ternalization, has been proposed as a correlate of diazepam tol- ␣ ␣ ␣ ␣ GABAA receptors containing the 1-, 2-, 3-, or 5-subunit in erance (Hutchinson et al., 1996; Itier et al., 1996; Primus et al., ␣ vivo, it has been shown that 1-GABAA receptors mediate the 1996; Ali and Olsen, 2001; Costa et al., 2001). Furthermore, subtle acute sedative (Rudolph et al., 1999; McKernan et al., 2000) and changes in the expression of GABAA receptor subunits were de- anticonvulsant (Rudolph et al., 1999) properties of diazepam, scribed notably in the cerebral cortex and hippocampus (Wu et ␣ whereas 2-GABAA receptors are the substrate for anxiolytic al., 1994; Impagnatiello et al., 1996; Pesold et al., 1997; Arnot et (Lo¨w et al., 2000) and muscle-relaxant activity, the latter also ␣ ␣ requiring 3- and 5-GABAA receptors (Crestani et al., 2001, Received March 23, 2004; revised June 1, 2004; accepted June 16, 2004. 2002). In view of such functional receptor specificity, we tested This work was supported by the Swiss National Science Foundation. We thank K. Lo¨w for the generation of the ␣ whether tolerance to a particular effect of diazepam is mediated 2(H101R) mice, D. Benke for help with autoradiography, and C. Sidler, H. Pocchetti, and E. Calame for technical assistance. via the same receptor subtype involved in the acute effect or *C.v.R. and M.T. contributed equally to this work. whether additional GABAA receptor subtypes are required for Correspondence should be addressed to Florence Crestani, Institute of Pharmacology and Toxicology, University neuronal plasticity leading to the development of tolerance with of Zu¨rich, Winterthurerstrasse 190, 8057 Zu¨rich, Switzerland. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.1067-04.2004 chronic diazepam treatment. Copyright © 2004 Society for Neuroscience 0270-6474/04/246785-06$15.00/0 In the present study, the contribution of specific GABAA re- 6786 • J. Neurosci., July 28, 2004 • 24(30):6785–6790 van Rijnsoever et al. • GABA Receptors and Diazepam Tolerance ceptor subtypes in the development of tolerance to the motor- Results depressant action of diazepam was examined using wild-type and Tolerance to the sedative action of diazepam in histidine–arginine point-mutated mice. Potential changes in point-mutated mice benzodiazepine binding sites were analyzed by autoradiography. To identify the diazepam-sensitive GABAA receptor subtypes im- Materials and Methods plicated in sedative tolerance, we examined the potential of his- ␣ tidine–arginine point-mutated mice to develop tolerance against Animals and drugs. Nine- to 11-week-old female wild-type, 1(H101R), ␣ ␣ ␣ Ͼ the motor-depressant action of diazepam during the course of a 2(H101R), 3(H123R), and 5(H105R) mice ( 10 backcrosses to 129/ SvJ background) were used (Rudolph et al., 1999; Lo¨w et al., 2000; Cres- chronic drug-treatment regimen. We focused on this behavioral tani et al., 2002). The sensitivity of GABAA receptors to diazepam is effect for two main reasons. First, the effectiveness of diazepam in conferred by the presence of a histidine residue at a conserved position in decreasing motor activity in rodents is considered as a valid be- the extracellular portions of the ␣ subunits (␣1-H101, ␣2-H101, ␣3-H126, havioral manifestation of its sedative properties (Vogel, 2002). ␣-H105). The histidine–arginine substitution in the GABA receptor ␣ -, ␣ A 1 Second, this drug effect is exclusively mediated by 1-GABAA ␣ -, ␣ -, or ␣ -subunit results in a marked reduction of the binding affinity ␣ 2 3 5 receptors, because it is abolished in 1(H101R) mice but unal- of the corresponding GABA receptor subtype to diazepam, as shown on A tered in ␣ (H101R), ␣ (H126R), and ␣ (H105R) mice (Rudolph recombinant receptors (Benson et al., 1998). Thus, each point-mutated 2 3 5 et al., 1999; Lo¨w et al., 2000; McKernan et al., 2000; Crestani et al., mouse line possesses a particular GABAA receptor subtype insensitive to allosteric modulation by diazepam, although its response to GABA is essen- 2002). tially preserved. Females were preferred to males because of the prominent Sedative tolerance to diazepam (i.e., the diminution of its intramale aggressive behavior inherent to the strain used as genetic back- motor-depressant action during chronic treatment) was first an- ␣ ground. They were reared in group-housed cages in the testing room under alyzed using wild-type and 1(H101R) mice. The different treat- reversed 12 hr light/dark conditions. Treatments and behavioral testing were ment conditions affected motor activity differently in wild-type performed during the dark phase. The Cantonal Veterinary Office of Zu¨rich mice (H ϭ 22.958; p Ͻ 0.001; n ϭ 6–7 mice per group). In approved all experimental procedures. Diazepam was from Hoffmann-La wild-type mice chronically treated with vehicle, the administra- Roche (Basel, Switzerland). tion of 10 mg/kg diazepam was followed by a marked decrease in Induction of sedative tolerance. Mice were subjected to daily injections Ͻ of diazepam (10 mg/kg at 10 A.M. and 5 mg/kg at 4 P.M., i.p.) for8din motor activity ( p 0.01 compared with Veh–Veh) (Fig. 1a). the home cage. On day 9, half of the mice were given vehicle (Diaz-Veh) This effect was comparable with that seen in mice that received a and the other half were given the morning dose of 10 mg/kg diazepam as single acute injection of diazepam. Mice chronically treated with the test dose. Control animals, which received the vehicle (0.3% Tween diazepam did not show a reduction in motor activity in response 80–saline solution) as chronic treatment, were distributed in two groups. to the test dose of diazepam and were indistinguishable from One group was treated with vehicle (Veh–Veh), and the other group was mice challenged with vehicle or from mice chronically treated treated with 10 mg/kg diazepam. A fifth group of mice, which served as ␣ with vehicle only (Fig. 1a). In 1(H101R) mice, neither a single control for the effects of repeated injections, received a single diazepam acute injection of diazepam (10 mg/kg) nor the chronic diazepam (10 mg/kg) injection as the test dose. Animals were left undisturbed to treatment altered the level of motor activity compared with the experience the drug effects in the home cage for 30 min.